1. Field of the Invention
The present invention relates generally to the fields of molecular virology and protein chemistry. More specifically, the present invention relates to the use of Human and Simian Immunodeficiency Virus (HIV/SIV) Gag proteins, or amino acid residues that mediate their packaging, as vehicles for delivery of proteins/peptides to virions or virus-like particles and uses thereof.
2. Description of the Related Art
Unlike simple retroviruses, human and simian immunodeficiency viruses (HIV/SIV) encode proteins in addition to Gag, Pol, and Env that are packaged into virus particles. These include the Vpr protein, present in all primate lentiviruses, and the Vpx protein, which is unique to the HIV-2/SIVSM/SIVMAC group of viruses. Since Vpr and Vpx are present in infectious virions, they have long been thought to play important roles early in the virus life cycle. Indeed, recent studies of HIV-1 have shown that Vpr has nucleophilic properties and that it facilitates, together with the matrix protein, nuclear transport of the viral preintegration complex in nondividing cells, such as the macrophage. Similarly, Vpx-deficient HIV-2 has been shown in exhibit delayed replication kinetics and to require 2-3 orders of magnitude more virus to produce and maintain a productive infection in peripheral blood mononuclear cells. Thus, both accessory proteins appear to be important for efficient replication and spread of HIV/SIV in primary target cells.
Incorporation of foreign proteins into retrovirus particles has previously been reported by fusion with gag. The yeast retrotransposon Ty1 was tested as a retrovirus assembly model to interfere with viral replication. (G. Natsoulis et al. Nature 1991, 352:632-5). More recently, the expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein was found to inhibit murine leukemia virus replication in tissue culture cells. The expression of Gag-staphylococcal nuclease reduces viral titer and diminishes viral infectivity to promote an anti-HIV strategy. (G. Schumann et al. J. Virol. 1996, 70:4329-37).
Lentiviral vectors, specifically those based on HIV-1, HIV-2 and SIV, have utility in gene therapy, due to their attractive property of stable integration into nondividing cell types (Naldini, L. et al. Science 1996, 272:263-267; Stewart, S. A. et al. J. Virol. 1997, 71:5579-5592; Zhang, J. et al. Science 1993, 259:234-238). The utility of lentiviral-based vector use for human therapy requires the development of a safe lentivral-based vector. HIV virion associated accessory proteins (Vpr and Vpx) have been shown to be useful as vehicles to deliver protein of both viral and non-viral origin into HIV particles (Liu, H. et al. J. Virol. 1995, 69:7630-7638; Liu, H. et al. J. Virol. 1997, 71:7704-7710; Wu, X. et al. J. Virol. 1994, 68:6161-6169; Wu, X. et al. EMBO Journal 1997, 16:5113-5122; Wu, X. et al. J. Virol. 1996, 70:3378-3384). We recently demonstrated that trans- RT and IN mimic cis- RT and IN (derived from Gag-Pol). The trans- RT and IN proteins effectively rescue the infectivity and replication of virions derived from RT-IN minus provirus through the complete life cycle (Liu, H. et al. J. Virol. 1997, 71:7704-7710; Wu, X. et al. J. Virol. 1994, 68:6161-6169). Moreover, these findings demonstrate that truncated Gag-Pol precursor polyprotein (Gag-Pro) support the formation of infectious particles when the functions of RT and IN are provided in trans. This finding demonstrated for the first time for a lentivirus that the full length Gag-Pol precursor is not required for the formation of infectious particles. Our data also show that trans Vpr-RT-IN, or Vpr-RT together with Vpr-IN are fully functional and support virus infectivity, integration of the proviral DNA, and replication (through one cycle) of RT defective, IN defective and RT-IN defective viruses (Liu, H. et al. J. Virol. 1997, 71:7704-7710; Wu, X et al. J. Virol. 1994, 68:6161-6169). It should also be noted that our data demonstrate that enzymatically active RT does not require Vpr for incorporation into virions (FIGS. 19A and B). RT can be incorporated into HIV-1 virions when expressed in trans even without its expression as a fusion partner of Vpr. These data demonstrate that the functions of these critical enzymes can be provided in trans, independent of their normal mechanism for expression and virion incorporation as components of the Gag-Pol precursor protein.
The prior art is deficient in the lack of effective means of delivering or targeting foreign, e.g., toxic proteins to virions. The present invention fulfills this longstanding need and desire in the art.
The present invention shows that Gag and/or Gag variants can be used as vehicles to target proteins of viral and non-viral origin into HIV/SIV virions. Gage gene fusions were constructed with bacterial staphylococcal nuclease (SN), chloramphenicol acetyl transferase (CAT) genes, green fluorescence protein (GFP), reverse transcriptase (RT), integrase (IN) and combinations thereof. Fusion proteins containing a Gag moiety should be packaged into HIV particles by expression in trans, to the native viral genome.
Gag fusion proteins were constructed and their abilities to package into HIV particles were demonstrated. The present invention shows that Gag fusion proteins were expressed in mammalian cells and were incorporated into HIV particles even in the presence of wild-type Gag proteins. The present invention further shows that virion incorporated Gag fusions remain infective in contrast to the prior art (G. Schuman et al. J. Virol. 1996, 70:4379-37). Thus, targeting heterologous Gag fusion proteins, including deleterious enzymes, to virions represents a new avenue toward anti-HIV drug discovery and gene therapy.
The invention shows that Gag proteins and variants thereof are operative as vehicles to deliver fully functional RT and IN in trans into lentiviral and retroviral particles, independently of their normal expression as components of the Gag-Pol precursor protein. Therefore this invention generates a novel packaging component (Gag-Pro), and a novel trans-enzymatic element that provides enzyme function for retroviral-based vectors. According to the present invention, the generation of potentially infectious/replicating retroviral forms (LTR-gag-pol-LTR) is decreased, since according to the present invention this requires recombination of at least three separate RNAs derived from the different plasmids: vector plasmid, packaging plasmid, a trans-enzyme expression plasmid and envelope plasmid, and as such is unlikely to occur. Virion Gag proteins are utilized in the present invention as vehicles to deliver the RT and IN proteins into lentiviral vectors, independently of Gag-Pol. As such, a xe2x80x9ctrans-lentiviralxe2x80x9d or xe2x80x9ctransretroviralxe2x80x9d vector is utilized for gene delivery, and gene therapy.
In one embodiment of the present invention, there is provided a composition of matter, comprising: DNA encoding a viral Gag protein fused to DNA encoding a virus inhibitory protein.
In another embodiment of the present invention, there is provided a composition of matter, comprising: DNA encoding a viral Gag protein truncate fused to DNA encoding a virus inhibitory protein.
In yet another embodiment of the present invention, there is provided a method of delivering a virus inhibitory molecule to a target in an animal, comprising the step of administering to said animal an effective amount of the composition of the present invention.
In still yet another embodiment of the present invention, there is provided a pharmaceutical composition, comprising a composition of the present invention and a pharmaceutically acceptable carrier.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.